Characterization of the lys2 gene of Penicillium chrysogenum encoding α-aminoadipic acid reductase

Abstract

A DNA fragment containing a gene homologous to LYS2 gene of Saccharomyces cerevisiae was cloned from a genomic DNA library of Penicillium chrysogenum AS-P-78. It encodes a protein of 1409 amino acids (Mr 154859) with strong similarity to the S. cerevisiae (49.9% identity) Schizosaccharomyces pombe (51.3% identity) and Candida albicans (48.12% identity) alpha-aminoadipate reductases and a lesser degree of identity to the amino acid-activating domains of the non-ribosomal peptide synthetases, including the alpha-aminoadipate-activating domain of the alpha-aminoadipyl-cysteinyl-valine synthetase of P. chrysogenum (12.4% identical amino acids). The lys2 gene contained one intron in the 5'-region and other in the 3'-region, as shown by comparing the nucleotide sequences of the cDNA and genomic DNA, and was transcribed as a 4.7-kb monocistronic mRNA. The lys2 gene was localized on chromosome III (7.5 Mb) in P. chrysogenum AS-P-78 and on chromosome IV (5.6 Mb) in strain P2, whereas the penicillin gene cluster is known to be located in chromosome I in both strains. The lys2-encoded protein is a member of the aminoacyladenylate-forming enzyme family with a reductase domain in its C-terminal region.

LYS2 gene and its mutation in Kluyveromyces lactis

“…The sequence of this enzyme appears to be highly conservative in the fungal kingdom, since the α-AA reductase of K. lactis was also similar to that of Pichia sorbitophila (identity of 57.2%; Accession No. AJ288950; Bleykasten-Grosshans et al, 2001), Candida albicans (55.7%; Accession No.U58 133; Suvarna et al, 1998) Bhattacherjee and Bhattacherjee, 1998), Penicillium chrysogenum (48.7%;Accession No.Y13 967;Casqueiro et al, 1998), Acremonium chrysogenum (46.8%; Accession No. AJ261064; Hijarrubia et al, 2001) and Neurospora crassa (48.9%; Accession No.AL389890; Schulte et al, unpublished).…”

“…Accordingly, it has been found that the sequences of α-AA reductases contain several distinctive domains in common, supposed to be involved in catalytic activities (Casqueiro et al, 1998;Hijarrubia et al, 2001): the adenylating domain shows homology with enzymes involved in amino acid activation via adenylation and is characterized by nine conserved motifs, including the AMP-binding and ATP-binding domains; the reductive domain, responsible for the reduction of the activated α-AA to its semialdehyde, showing homologies with dehydrogenases; a region is involved in phosphopantetheinylation, a post-translational modification mediated by the product of LYS5 gene and required for the activation of the LYS2-encoded α-AA reductase (Ehmann et al, 1999). Alignments of the seven yeast sequences showed that these functional regions are extremely well conserved (data not shown).…”

Novel Posttranslational Activation of the LYS2- Encoded α-Aminoadipate Reductase for Biosynthesis of Lysine and Site-Directed Mutational Analysis of Conserved Amino Acid Residues in the Activation Domain of Candida albicans

“…The open reading frame of the LYS2 gene consists of 4,176 nucleotides (nt) encoding 1,392 amino acid residues in S. cerevisiae (1,25), 4,173 nt encoding 1,391 amino acid residues in C. albicans (21,34), 4,330 nt encoding 1,409 amino acid residues in Penicillium chrysogenum (11), and 4,245 nt encoding 1,415 amino acid residues in Schizosaccharomyces pombe (8). These genes and the encoded approximately 150-kDa proteins exhibit more than 60% identity at the nucleotide level and 55% identity at the amino acid level.…”

“…These genes and the encoded approximately 150-kDa proteins exhibit more than 60% identity at the nucleotide level and 55% identity at the amino acid level. Additionally, there exist several highly conserved core sequences and functional domains in the Lys2p which correspond to those in the nonribosomal peptide synthetases such as ␣-aminoadipyl-L-cysteinyl-D-valine synthetase for penicillin biosynthesis (8,11,18,34). Computer analysis also revealed the presence of a highly conserved phosphopantetheinylation domain (LGGHSI, amino acid residues 880 to 885) in Lys2p (34).…”

Conversion of Pipecolic Acid into Lysine in Penicillium chrysogenum Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the lys7 Gene Encoding Saccharopine Reductase

“…␣-Aminoadipic acid has a key function in penicillin biosynthesis, since addition of exogenous ␣-aminoadipic acid (21) or genetic modifications that increase the internal ␣-aminoadipic acid pool (11,22,23) lead to a stimulation of the rate of penicillin biosynthesis. ␣-Aminoadipic acid is converted into piperideine-6-carboxylic acid (P6C) by ␣-aminoadipate reductase (9), and the piperideine-6-carboxylate may be reduced to pipecolic acid.…”